A selenium-containing soluble composite microneedle and its preparation method and application

Abstract

The present invention discloses a selenium-containing soluble composite microneedle and its preparation method and application, belonging to the technical fields of biomaterials and biomedicine. The microneedle forming matrix is selenium hyaluronate, loaded with drug-loaded tetrahedral framework nucleic acid, and the base part is polyvinyl alcohol. The preparation method includes: Step 1, preparation of selenium hyaluronate; Step 2, preparation and drug loading of tetrahedral framework nucleic acid; Step 3, preparation of selenium-containing and drug-loaded microneedle patches. The present invention synthesizes selenium hyaluronate by a specific chemical method, uses selenium hyaluronate as the base, loads drug-loaded tetrahedral framework nucleic acid, and utilizes the advantages of high permeability, good biocompatibility, and editability of tetrahedral framework nucleic acid to improve the sustained and controlled release ability of microneedles, realize the efficient transdermal delivery of drugs, and reduce the pain and discomfort that may be brought by traditional drug delivery methods. It provides a new and effective drug delivery system in the field of psoriasis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomaterials and biomedicine, and particularly relates to a selenium-containing soluble composite microneedle for psoriasis treatment, and a preparation method and application thereof. Background Art

[0002] Psoriasis is a chronic inflammatory skin disease, and its treatment methods have made significant progress in recent years. Current treatment methods mainly include topical therapy, phototherapy, systemic immunomodulators, and biologics. These methods aim to relieve symptoms and improve the quality of life of patients. However, there are still some challenges, such as adverse reactions, treatment resistance, high costs, and differences in individual responses. In recent years, with the in-depth study of the pathogenesis of psoriasis, the research and development of new drugs have developed rapidly. Traditional treatment methods include drug therapy, phototherapy, and biologics, etc. Although they can relieve symptoms to a certain extent, there are problems such as limited efficacy, large side effects, and high recurrence rates.

[0003] Microneedle therapy provides a new treatment idea. It creates tiny channels on the skin surface by using fine needles to promote drug penetration and skin repair. As an innovative physical therapy method, microneedle therapy shows unique advantages and application prospects in the treatment field of psoriasis. Patent CN202110836016.6 uses a pH-responsive polymer micelle coated with human keratinocyte membrane to construct an epidermis-active targeted drug delivery system, and uses microneedle technology to assist the transdermal delivery of the anti-inflammatory drug shikonin, prolonging the action time. Patent CN202010809588.0 discloses the preparation of soluble microneedles that can effectively achieve the programmed release of psoriasis treatment drugs, leaving sustained-release particles in the skin to maintain a long-term effective drug treatment effect. However, in the currently disclosed patents, microneedles only serve as a drug delivery system, and the microneedles themselves lack functionalization.

[0004] Patients with psoriasis have a higher level of oxidative stress in their bodies. Oxidative stress refers to the excessive production of free radicals or the decline in antioxidant capacity in the body, resulting in damage to cells and tissues. The levels of free radicals in the skin and blood of psoriasis patients increase, and these free radicals can damage skin cells and exacerbate the inflammatory response. Selenium, as an essential trace element, plays an important role in the treatment of psoriasis, and its mechanism mainly involves three aspects: antioxidant, immunomodulation, and cell protection. Selenium can enhance the function of the body's endogenous antioxidant system. By increasing the activities of antioxidant enzymes such as glutathione peroxidase, it can effectively scavenge free radicals and peroxides, reducing the damage of oxidative stress to the skin. The immunomodulatory effect of selenium is reflected in its ability to balance the imbalance of Th1 / Th2 cell subsets, inhibit the release of inflammatory factors such as IFN-γ triggered by the overactivation of Th1 cells, and at the same time promote the production of anti-inflammatory cytokines such as IL-4, thereby reducing the skin inflammation of psoriasis patients. In addition, selenium can directly act on skin cells, protect cell membranes from oxidative damage, maintain the integrity of cell structure and function, help inhibit the abnormal proliferation and differentiation of epidermal cells, and improve the skin lesion symptoms of psoriasis patients. Currently, there is no report on the application of selenium-containing microneedles in the treatment of psoriasis. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a selenium-containing soluble composite microneedle, its preparation method, and its application in the treatment of psoriasis. The selenium-containing soluble composite microneedle provided by the present invention can exert an antioxidant effect, effectively scavenge reactive oxygen species, regulate the immune microenvironment, reduce inflammation, and control the chronic inflammatory state of psoriasis. At the same time, it can carry drugs and nanoformulations, and has a good therapeutic effect on psoriasis. At the same time, to improve the sustained and controlled release effect of drugs, the present invention also provides tetrahedral framework nucleic acid (tFNA) as a drug delivery carrier to achieve the purpose of sustained and controlled release of drugs and realize the efficient transdermal delivery of drugs.

[0006] A selenium-containing soluble composite microneedle of the present invention, the microneedle forming matrix is seleniumized hyaluronic acid, loaded with drug-carrying tetrahedral framework nucleic acid, and the base part is polyvinyl alcohol with good biocompatibility.

[0007] The present invention also provides a preparation method for the above selenium-containing soluble composite microneedle, including the following steps:

[0008] Step 1: Preparation of seleniumized hyaluronic acid:

[0009] 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to the hyaluronic acid solution and stirred for 0.5 h to 24 h for activation. A selenium-containing small molecule compound was continuously added to the solution, and the reaction was carried out in the dark for 8 h to 48 h under the protection of an inert gas. After the reaction was completed, dialysis was performed 2 to 3 times with distilled water using a dialysis bag (Mw 3000), and freeze-drying was carried out at -60 °C for 40 h to 48 h to obtain selenium-containing hyaluronic acid.

[0010] Step 2: Preparation of tetrahedral framework nucleic acid and drug loading:

[0011] (1) Sequence design: Four single-stranded DNAs were designed, each strand containing three complementary regions and one non-complementary region. The length of the complementary region was 20 - 30 bases to ensure the formation of stable double strands, and the length of the non-complementary region was 5 - 10 bases to form the vertices of the tetrahedron;

[0012] (2) Annealing and assembly: The single-stranded DNA was dissolved in a buffer containing 10 mM Tris-HCl and 50 mM MgCl2, and then the mixture was maintained at 80 °C to 105 °C for 5 min to 15 min, and slowly cooled to 2 °C to 8 °C and maintained for 10 min to 60 min to allow the DNA to self-assemble into a tetrahedral framework structure. The product was stored at 4 °C;

[0013] (3) Drug loading: A DMSO solution of the hydrophobic drug was slowly added dropwise to the prepared tetrahedral framework nucleic acid solution, and the mixture was stirred in the dark for 1 h to 24 h. Ultrafiltration tubes (10 kDa - 50 kDa) were used for centrifugation to remove the unloaded drug, and the drug-loaded tetrahedral framework nucleic acid was obtained.

[0014] Step 3: Preparation of selenium-containing and drug-loaded microneedle patches:

[0015] (1) In a laminar flow hood, the selenium-containing hyaluronic acid obtained in Step 1 was weighed into a sterile container, mixed with the drug-loaded tetrahedral framework nucleic acid obtained in Step 2, and ultrasonicated with ultrapure water to dissolve and prepare a solution as the needle layer gel solution;

[0016] (2) A polyvinyl alcohol (PVA) solution prepared by dissolving PVA in pure water was used as the backing layer gel solution;

[0017] (3) Both the needle layer gel solution and the backing layer gel solution were placed in a centrifuge for degassing treatment;

[0018] (4) The degassed needle layer gel solution was added to the microneedle holes of the microneedle mold using a syringe, placed in a vacuum drying oven for vacuum pumping, and the mold was centrifuged to scrape off the excess colloid on the surface;

[0019] (5) Re-add the needle layer gel solution, and repeat steps (4)-(5) until each needle hole is filled, then place it in a vacuum drying oven or a constant temperature and humidity chamber for drying;

[0020] (6) Add the backing layer gel solution to the grooves of the microneedle mold as the backing layer of the microneedles, then place it at 37°C for drying, and demold to obtain the microneedle patch.

[0021] The preparation method of the above-mentioned selenium-containing soluble composite microneedles, wherein:

[0022] Further, in the step 1, the solvent of the hyaluronic acid solution is selected from water, DMSO, DMF or a mixed solution of any ratio thereof.

[0023] Further, in the step 1, the selenium-containing small molecule compound is selected from selenocystamine, Se-(methyl)selenocysteine, selenomethionine, selenocysteine and their corresponding hydrochlorides. The molar ratio of hyaluronic acid to the selenium-containing small molecule compound is (0.1-5):1. The hyaluronic acid is selected from one of those with a molecular weight of 3 kDa-10 kDa, 10 kDa-100 kDa, 200 kDa-400 kDa, 1100 kDa-1300 kDa.

[0024] Further, in the step 2(1), the four complementary single-stranded DNA sequences (5′→3′) are as shown in SEQ ID NO.1-4.

[0025] Further, in the step 2(2), the molar ratio of the four single-stranded DNAs is 1:1:1:1. The pH value of the buffer solution is 7.5-8.5, and the optimal value is 8.0. The heating temperature of the mixture is 80°C-105°C, the optimal value is 95°C, the heating time is 5 min-15 min, the optimal value is 10 min. Slowly cool down to 2°C-8°C, the optimal value is 4°C, the cooling rate is 1°C / min-3°C / min, and the maintenance time is 10 min-60 min, the optimal value is 30 min.

[0026] Further, in the step 2(3), the hydrophobic drug mainly refers to a drug containing an aromatic ring that can treat psoriasis, including but not limited to methotrexate, tofacitinib, apremilast, tacrolimus, tazarotene, rapamycin, shikonin.

[0027] Further, in the step 2(3), the molar ratio of the drug to the tetrahedral framework nucleic acid is (100-1000):1. The light-shielding stirring temperature is 4°C-25°C, the optimal value is 4°C, the stirring time is 1 h-24 h, the optimal value is 6 h. The cut-off molecular weight of the ultrafiltration tube is 10 kDa-50 kDa, and the appropriate size is selected according to the molecular weight of the drug.

[0028] Further, in the step 3(1), the concentration of the seleniumized hyaluronic acid is 5% - 50% (w / v). The ratio of the drug in the drug-loaded tetrahedral framework nucleic acid to the seleniumized hyaluronic acid is (1 - 10):100.

[0029] Further, in the step 3(2), the concentration of the polyvinyl alcohol is 5% - 30% (w / v).

[0030] Further, in the step 3(3), the rotation speed for centrifugal degassing is 2000 rpm - 5000 rpm, and the optimal speed is 3000 rpm. The degassing time is 2 min - 3 min.

[0031] Further, in the step 3(4), the vacuum pressure is 0.09 MPa - 0.1 MPa, and the placement time is 2 min - 3 min. The rotation speed for centrifugal adjustment is 2000 rpm - 5000 pm, and the optimal speed is 4000 rpm. The centrifugation time is 2 min - 3 min.

[0032] Further, in the step 3(5), the drying time is 6 h - 24 h.

[0033] Further, in the step 3(6), the drying can be carried out in a vacuum drying oven or in a constant temperature and humidity box, and the drying time is 24 h - 72 h.

[0034] Application of a selenium-containing soluble composite microneedle disclosed by the present invention in the preparation of a therapeutic administration patch for psoriasis.

[0035] The selenium-containing soluble composite microneedle of the present invention can also be applied to the preparation of drugs for treating other oxidative stress skin diseases, such as ulcers and infected wound healing.

[0036] Key points of the present invention:

[0037] The present invention synthesizes seleniumized hyaluronic acid through a specific chemical method and prepares microneedles using seleniumized hyaluronic acid as the substrate. Compared with traditional microneedle materials, the microneedles prepared by the present invention not only have the function of drug delivery but also have significant antioxidant ability, can effectively scavenge reactive oxygen species, and improve the immune environment. At the same time, by utilizing the advantages of tetrahedral framework nucleic acid such as high permeability, good biocompatibility, editable property, high stability, and easy preparation, the sustained and controlled release ability of the microneedles is improved, realizing efficient transdermal drug delivery and reducing the pain and discomfort that may be brought by traditional drug delivery methods.

[0038] Beneficial effects of the present invention:

[0039] By combining selenium - modified hyaluronic acid and tetrahedral framework nucleic acid, the present invention improves the drug - loading capacity and delivery efficiency, enabling the drug to reach the target site more effectively. The antioxidant and immunomodulatory effects of selenium - modified hyaluronic acid help alleviate the inflammatory response of psoriasis and improve the treatment effect. The soluble microneedle technology reduces the pain and discomfort that may be brought by traditional drug - delivery methods and improves patient compliance. The painless transdermal drug - delivery system improves patient comfort and makes the treatment process more user - friendly. The advantages of tetrahedral framework nucleic acid, such as high permeability, good biocompatibility, editability, high stability, and easy preparation, improve the drug - loading capacity and delivery efficiency.

[0040] The technical solution of the present invention combines a variety of advanced technologies, providing a new and effective drug - delivery system in the field of psoriasis treatment, which is expected to improve the existing treatment methods, enhance the treatment effect, reduce side effects, and improve the quality of life of patients. Brief Description of the Drawings

[0041] Figure 1 It is the Fourier transform infrared spectrum of hyaluronic acid and selenium - modified hyaluronic acid in the embodiment of the present invention;

[0042] Figure 2 It is the overall scanning electron microscope image (a) and local magnified image (b) of the microneedles in the embodiment of the present invention;

[0043] Figure 3 It is the elemental mapping image of the microneedles in the embodiment of the present invention;

[0044] Figure 4 It is the mechanical properties and in vitro transdermal release effect of the microneedle patch in the embodiment of the present invention; where (a) is the mechanical properties of selenium - containing composite microneedles and hyaluronic acid matrix microneedles, and (b) is the in vitro transdermal release of shikonin from the microneedle patch;

[0045] Figure 5 It is the cell uptake effect of shikonin in the embodiment of the present invention (red: shikonin, blue: cell nucleus, green: cell membrane);

[0046] Figure 6 It is the effect of SeHA on scavenging reactive oxygen species in RAW 264.7 cells and HacaT cells under oxidative stress in the embodiment of the present invention; where (a) is the effect of SeHA on scavenging reactive oxygen species in RAW 264.7 cells under oxidative stress, and (b) is the effect of SeHA on scavenging reactive oxygen species in HacaT cells under oxidative stress;

[0047] Figure 7Experimental results of SeHA scavenging reactive oxygen species in the embodiments of the present invention; among them, (a) shows the protective effect of SeHA on RAW 264.7 cells under oxidative stress in the MTT assay, and (b) shows the protective effect of SeHA on HaCaT cells under oxidative stress in the MTT assay;

[0048] Figure 8 Detection results of IL-6 and TNF-α levels in the quantitative H2O2-induced oxidative stress RAW cell model in the embodiments of the present invention; among them, (a) shows the detection result of the IL-6 level, and (b) shows the detection result of the TNF-α level;

[0049] Figure 9 Treatment effects of imiquimod-induced psoriasis mice in different treatment groups in the embodiments of the present invention; among them, (a) shows the skin surface after treatment with different treatment groups, and (b) shows the PASI score of psoriatic skin. Detailed implementation manners

[0050] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0051] Unless otherwise specified, the instruments, reagents and materials used in the present invention are all commercially available.

[0052] Example 1

[0053] Preparation of selenium hyaluronic acid, including:

[0054] (1) Selenocystamine-modified hyaluronic acid:

[0055] 100 mg of hyaluronic acid (10 - 100 kDa) is dissolved in 50 mL of DMSO, 251 mg of 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 151 mg of N-hydroxysuccinimide (NHS) are added, and the molar ratio of the two is 1:1. Stir at room temperature for 6 h. Selenocystamine dihydrochloride is slowly added to the above solution, and a dark reaction is carried out for 48 h under the protection of an inert gas. After the reaction is completed, dialysis is carried out 3 times with distilled water using a dialysis bag (Mw 3000), and freeze-drying is carried out at -60 °C for 48 h to obtain selenocystamine-modified hyaluronic acid. The results are shown in Figure 1 Fourier transform infrared spectroscopy, and the infrared spectrum shows the appearance of C-Se (771 cm -1 ) and Se-Se (615 cm -1 ) peaks, which confirm the diselenide and prove that selenocysteine is successfully modified on hyaluronic acid.

[0056] (2) Se-(methyl)selenocysteine-modified hyaluronic acid:

[0057] 50 mg of hyaluronic acid (3 - 10 kDa) was dissolved in 50 mL of DMSO / water (1:1, v / v). 126 mg of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC·HCl) and 76 mg of N-hydroxysuccinimide (NHS) were added, and their molar ratio was 1:1. The mixture was stirred at room temperature for 8 h. Se-(methyl)selenocysteine hydrochloride was slowly added to the above solution, and the reaction was carried out in the dark under the protection of inert gas for 48 h. After the reaction was completed, dialysis was performed 3 times with distilled water using a dialysis bag (Mw 3000), and freeze-drying was carried out at -60 °C for 48 h to obtain Se-(methyl)selenocysteine-modified hyaluronic acid.

[0058] Example 2

[0059] The preparation of tetrahedral framework nucleic acid has the following steps:

[0060] (1) Sequence design: Four single-stranded DNAs were designed, and each strand contained three complementary regions and one non-complementary region. The length of the complementary regions was 20 - 30 bases to ensure the formation of stable double strands, and the length of the non-complementary region was 5 - 10 bases to form the vertices of the tetrahedron. The four complementary single-stranded DNA sequences (5′→3′) designed in the present invention are shown in SEQ ID NO.1 - 4.

[0061] (2) Annealing and assembly: The four single-stranded DNAs (final concentration of 10000 nM) were dissolved in a buffer containing 10 mM Tris-HCl and 50 mM MgCl2, and the molar ratio of the four single-stranded DNAs was 1:1:1:1. The pH value of the buffer was 8.0. Then the mixture was maintained at 95 °C for 10 min, and then slowly cooled to 4 °C and maintained for 30 min, with a cooling rate of 2 °C / min, so that the DNA self-assembled to form a tetrahedral framework structure. The obtained tetrahedral framework nucleic acid product was stored at 4 °C for further use.

[0062] Example 3

[0063] Drug loading of tetrahedral framework nucleic acid:

[0064] 50 mL of shikonin DMSO solution (500 mM) was slowly added dropwise to the tetrahedral framework nucleic acid solution prepared in Example 2, and the mixture was stirred and mixed evenly in the dark at 4 °C for 6 h. The unloaded drug was removed by centrifugation using an ultrafiltration tube (30 kDa) to obtain the drug-loaded tetrahedral framework nucleic acid.

[0065] Example 4

[0066] The preparation of selenium-containing composite microneedles loaded with tetrahedral framework nucleic acid is as follows:

[0067] (1) In a laminar flow hood, weigh 0.8 g of the selenium - cysteamine - modified hyaluronic acid prepared in Example 1, and add it to the drug - loaded tetrahedral framework nucleic acid solution prepared in Example 3 in 3 batches. Ultrasonically dissolve it to obtain the needle - layer gel solution.

[0068] (2) Weigh polyvinyl alcohol (PVA) and dissolve it in pure water to make a 20% polyvinyl alcohol solution as the backing - layer gel solution.

[0069] (3) Place both the needle - layer gel solution and the backing - layer gel solution in a centrifuge at 3000 rpm for 3 min for degassing treatment.

[0070] (4) Use a syringe to add the degassed needle - layer gel solution to the micro - needle holes of the micro - needle mold. Place it in a vacuum drying oven and evacuate (0.09 MPa - 0.1 MPa) and maintain for 2 min. Then centrifuge the mold (4000 rpm, 3 min) and scrape off the excess colloid on the surface.

[0071] (5) Re - add the needle - layer gel solution, and repeat steps (4) - (5) 1 - 2 times until each needle hole is filled. Then place it in a vacuum drying oven and statically dry at 37 °C for 24 h.

[0072] (6) Add the backing - layer gel solution to the groove of the micro - needle mold as the backing layer of the micro - needles. Then place it in a static drying at 37 °C for 72 h, and demold to obtain the selenium - containing composite micro - needle patch loaded with tetrahedral framework nucleic acid.

[0073] Characterization and performance: The size of the micro - needle patch prepared in this example is 14.5×14.5 mm, which consists of a regular square pyramid formed by a 15×15 array. The bottom side length of each needle is 360 μm and the height is 800 μm, and the tip - to - tip distance is 720 μm.

[0074] The micro - needle patch prepared in this example was characterized by a scanning electron microscope, and the results are shown in Figure 2 . It can be seen from the scanning electron microscope that the micro - needles have a complete morphology and are regular square pyramids. At the same time, Figure 3 the elemental mapping image shows the presence of C, N, O, and Se elements at the tip, indicating that selenium - modified hyaluronic acid has been successfully integrated into the micro - needles.

[0075] The mechanical properties of the micro - needle patch prepared in this example were tested by an electronic universal material testing machine, and the results are shown in Figure 4 (a). The force exerted by the micro - needle patch at a displacement of 800 μm is greater than 0.3 N / needle, having sufficient characteristics to penetrate the skin. And compared with the traditional hyaluronic acid - based micro - needles (blue HA), the selenium - containing composite micro - needles (red tFNAs / shikonin - SeHA) prepared in this example have better mechanical strength.

[0076] The micro - needle patch prepared in this example was studied for its sustained - release effect through an in - vitro transdermal release experiment, and the results are shown in Figure 4 (b). As time goes by, the cumulative release percentage of shikonin gradually increases. Within the first 12 h, the release rate of shikonin is relatively fast, and the cumulative release percentage rapidly increases to about 40%. Subsequently, the release rate gradually slows down, and the cumulative release percentage reaches about 80% at 72 h. These results indicate that the prepared selenium - containing composite micro - needle patch can effectively release drugs in an in - vitro simulated in - vivo environment.

[0077] In summary, the selenium - containing composite micro - needle patch described in the present invention has good mechanical properties and drug - release characteristics, achieving the purpose of sustained - release and controlled - release of drugs, and can realize efficient transdermal delivery of drugs.

[0078] Application Example 1

[0079] Test the effect of tetrahedral framework nucleic acid (tFNA) on promoting the uptake of shikonin by cells:

[0080] HacaT cells were treated with shikonin or drug - loaded tetrahedral framework nucleic acid (tFNAs / shikonin) prepared in Example 3 for 8 h, and then a confocal microscope was used to examine how tFNA effectively helps shikonin penetrate HacaT cells. As Figure 5 shown, in the presence of tFNA, the fluorescence of tFNAs / shikonin is significantly stronger than that of shikonin (red), indicating that more shikonin enters the cells.

[0081] Application Example 2

[0082] Test the effect of selenium - modified hyaluronic acid on scavenging intracellular reactive oxygen species and reducing intracellular inflammatory factors:

[0083] Evaluated by reactive oxygen species probes and MTT experiments, the selenium - cysteamine - modified hyaluronic acid (SeHA) prepared in Example 1 shows a protective effect on RAW264.7 cells and HacaT cells experiencing oxidative stress.

[0084] Under the stress condition induced by 100 μM H2O2, a model of high intracellular reactive oxygen species levels in Hacat and RAW 264.7 cells was obtained. Through 2',7' - dichlorofluorescein (DCF) staining, the changes in DCF fluorescence intensity in different treatment groups could be observed. The results showed that 400 μM SeHA could significantly reduce the increase in DCF fluorescence intensity, while 400 μM HA (without Se - Se bonds) in the control group did not. The results are shown in Figure 6Furthermore, the MTT assay was used to verify the protective cell viability of SeHA against the two cell types. The cell viability of the SeHA treatment group was significantly higher than that of the HA treatment group, indicating that SeHA has a protective effect on cells, further confirming effective reactive oxygen species elimination. The results are shown in Figure 7 .

[0085] In addition, we also detected the antioxidant effect of SeHA in H2O2-induced RAW 264.7 cells by measuring the levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the cell supernatant. The results are shown in Figure 8 , SeHA significantly reduced the production of H2O2-induced IL-6 and TNF-α, and there was no obvious effect in the HA treatment group, indicating that SeHA can not only scavenge reactive oxygen species, but also reduce the production of inflammatory factors induced by oxidative stress.

[0086] In summary, the selenium-cystamine-modified hyaluronic acid prepared in Example 1 can effectively scavenge ROS induced by H2O2 and significantly reduce the level of intracellular inflammatory factors, indicating the application potential of selenium hyaluronic acid as a potential antioxidant in cell protection.

[0087] Application Example 3

[0088] Anti-psoriatic effect of the tetrahedral framework nucleic acid-containing selenium composite microneedles prepared in Example 4:

[0089] Animal model establishment: 1) Twenty-five 6-8-week-old C58BL / 6 mice were selected and randomly divided into 5 groups: control group, imiquimod treatment group (model group), shikonin cream group (SHI) group, selenium hyaluronic acid microneedle patch (SeHA) group, and tetrahedral framework nucleic acid-loaded shikonin-containing selenium composite microneedle patch (tFNAs / shikonin-SeHA) group. There were 5 mice in each group, and the backs of the mice were depilated; 2) The control group was not treated; the mice in the imiquimod model group were smeared with 62.5 mg of imiquimod cream daily; the mice in the shikonin cream group were smeared with 62.5 mg of imiquimod cream daily, and then smeared with 62.5 mg of shikonin cream dissolved in petrolatum daily; the mice in the SeHA group were smeared with 62.5 mg of imiquimod cream daily, and then given a microneedle patch; the mice in the tFNAs / shikonin-SeHA group were smeared with 62.5 mg of imiquimod cream daily, and then given a microneedle patch for 7 consecutive days; 3) On the 8th day, the mice were euthanized, and the back skin of the mice was collected for subsequent detection.

[0090] Perform PASI scoring on mice: Take pictures of the back skin of mice daily to observe local erythema, scaling, and skin thickening. Use the Modified psoriasis severity index score (PASI) to score the erythema, scaling, and thickening of the skin lesions in mice as follows: 0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, extremely severe. The clinical photo results of mice in different groups after treatment are shown in Figure 9 (a), and the PASI scores of mice in different groups are shown in Figure 9 (b), which are the erythema score, scaled score, thickness score, and total PASI score, respectively.

[0091] Experimental results: As shown by Figure 9 , imiquimod cream induced obvious psoriasis symptoms such as erythema, scaling, and epidermal thickening in mice, successfully inducing a psoriasis mouse model. No erythema, scaling, or thickening occurred in the skin of the normal group during the 1d - 7d experimental period. However, varying degrees of skin lesions appeared in other experimental groups. Obvious erythema symptoms began to appear in the model group within 2 days of modeling, accompanied by mild scaling and keratin thickening. Subsequently, the PASI score increased rapidly and reached the maximum value on the 6th day. Topical application of shikonin, selenium hyaluronic acid microneedle patches, and tetrahedral framework nucleic acid-loaded shikonin selenium-containing composite microneedle patches to imiquimod-induced skin inflammation mice could all alleviate manifestations such as epidermal thickening, scaling, and erythema in psoriasis model mice. As shown by Figure 9 , both the appearance and clinical scores of mice in the shikonin cream group were better than those of mice in the SeHA group, proving that shikonin has a certain therapeutic effect, but the treatment effect of mice in the tFNAs / shikonin-SeHA group was better.

Claims

1. A preparation method of a selenium-containing soluble composite microneedle, characterized in that, Proceed as follows: Step 1. Preparation of selenium - modified hyaluronic acid: Add 1 - [3 - dimethylaminopropyl] - 3 - ethylcarbodiimide and N - hydroxysuccinimide to the hyaluronic acid solution, stir for 0.5 h to 24 h for activation. Then continue to add a selenium - containing small - molecule compound to the solution, and carry out a dark reaction for 8 h to 48 h under the protection of inert gas. After the reaction is completed, dialyze 2 - 3 times, and obtain selenium - modified hyaluronic acid by freeze - drying at - 60 °C. The molar ratio of hyaluronic acid to the selenium - containing small - molecule compound is (0.1 - 5):1; Step 2. Preparation of tetrahedral framework nucleic acid and drug loading: Design four complementary single - strand DNA sequences as shown in SEQ ID NO.1 - 4; anneal and assemble to make the DNA self - assemble into a tetrahedral framework nucleic acid, and store the product at 4 °C. Slowly drop the hydrophobic drug solution into the tetrahedral framework nucleic acid solution, stir evenly in the dark for 1 h to 24 h, and centrifuge with an ultrafiltration tube to remove the unloaded drug, obtaining the drug - loaded tetrahedral framework nucleic acid. The molar ratio of the drug to the tetrahedral framework nucleic acid is (100 - 1000):1; the hydrophobic drug is shikonin; Step 3. Preparation of selenium - containing and drug - loaded microneedles: (1) Weigh the selenium - modified hyaluronic acid obtained in Step 1 in a sterile container, mix it with the drug - loaded tetrahedral framework nucleic acid obtained in Step 2, add ultrapure water and dissolve it by ultrasonic to form a solution, which is used as the needle - layer gel solution. The concentration of the selenium - modified hyaluronic acid is 5% - 50%; the ratio of shikonin in the drug - loaded tetrahedral framework nucleic acid to the selenium - modified hyaluronic acid is (1 - 10):100; (2) Weigh polyvinyl alcohol and dissolve it in pure water to make a 5% - 30% polyvinyl alcohol solution as the backing - layer gel solution; (3) Place the needle - layer gel solution and the backing - layer gel solution in a centrifuge for degassing treatment; (4) Use a syringe to add the degassed needle - layer gel solution into the microneedle holes of the microneedle mold, place it in a vacuum drying oven to evacuate, centrifuge the mold, and scrape off the excess colloid on the surface; (5) Re - add the needle - layer gel solution; (6) Repeat steps (4) - (5) until each needle hole is filled, and then dry; (7) Add the degassed backing - layer gel solution to the groove of the microneedle mold as the backing layer of the microneedle, and then dry it at 37 °C and demold to obtain the microneedles.

2. The preparation method of a selenium-containing soluble composite microneedle according to claim 1, characterized in that, In Step 1, the selenium - containing small - molecule compound is selected from selenocystamine, methylselenocysteine, selenomethionine, and selenocysteine.

3. The preparation method of a selenium-containing soluble composite microneedle according to claim 1, characterized in that, In Step 3 (3), the rotation speed for centrifugal degassing is 2000 rpm to 5000 rpm, and the degassing time is 2 min to 3 min; in Step 3 (4), the vacuum pressure is 0.09 MPa to 0.1 MPa, and the placement time is 2 min to 3 min; the centrifugal adjustment rotation speed is 2000 rpm to 5000 pm, and the centrifugal time is 2 min to 3 min.

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